Interactions of an Artificial Zinc Finger Protein with Cd(II) and Hg(II): Competition and Metal and DNA Binding

Cys2His2 zinc finger proteins are important for living organisms, as they—among other functions—specifically recognise DNA when Zn(II) is coordinated to the proteins, stabilising their ββα secondary structure. Therefore, competition with other metal ions may alter their original function. Toxic metal ions such as Cd(II) or Hg(II) might be especially dangerous because of their similar chemical properties to Zn(II). Most competition studies carried out so far have involved small zinc finger peptides. Therefore, we have investigated the interactions of toxic metal ions with a zinc finger proteins consisting of three finger units and the consequences on the DNA binding properties of the protein. Binding of one Cd(II) per finger subunit of the protein was shown by circular dichroism spectroscopy, fluorimetry and electrospray ionisation mass spectrometry. Cd(II) stabilised a similar secondary structure to that of the Zn(II)-bound protein but with a slightly lower affinity. In contrast, Hg(II) could displace Zn(II) quantitatively (logβ′ ≥ 16.7), demolishing the secondary structure, and further Hg(II) binding was also observed. Based on electrophoretic gel mobility shift assays, the Cd(II)-bound zinc finger protein could recognise the specific DNA target sequence similarly to the Zn(II)-loaded form but with a ~0.6 log units lower stability constant, while Hg(II) could destroy DNA binding completely

Módszerfejlesztés fehérje-glikoziláció analízisére = Method development for protein glycosylation analysis

Az extracelluláris glikoziláció tanulmányozása némiképp elhanyagolt kutatási terület. Ennek az egyik oka az elképesztő heterogenitás: egy adott pozíció hol módosított, hol nem, és számtalan különböző cukor-szerkezetet viselhet, így a glikopeptidek többnyire szubsztöchiometrikus mennyiségben fordulnak elő. Ráadásul a poszt-transzlációs módosítások vizsgálatára általában használatos tömegspektrometria is nehezebben boldogul a glikopeptidekkel. Mi szérum-fehérjék Ser és Thr oldalláncát módosító gyakori és egyszerű cukrok vizsgálatára fókuszáltunk. Marhaszérummal dolgoztunk. Egy dúsítási eljárást dolgoztunk ki egy cukorkötő-fehérje (lektin) segítségével. A glikopeptid-elegyet egy új MS/MS technika: elektron-transzfer disszociáció (ETD) segítségével analizáltuk. Ennek sikeréhez az adatbázis-lekereső szoftvert is optimalizálni kellett az ETD adatokhoz. Kutatásunk során kb. 40 új glikozilációs helyet azonosítottunk. Ennyit eddig senkinek nem sikerült egyetlen kísérlet-sorozatból. Sejten belül is előfordul O-glikoziláció, egyetlen GlcNAc kerül a Ser/Thr oldalláncokra, regulációs és jelátviteli szerepe van. Bár biológiai szempontból nagyon eltér az extracelluláris rokonságtól, hasonló analitikai kihívást jelent. Erre a módosításra is kidolgoztunk egy dúsítási eljárást. | Studying extracellular glycosylation is a somewhat neglected research area. Partly because the incredible heterogeneity glycoproteins feature both in site occupancy and in the number of different sugar structures modifying the same site. Thus, glycopeptides almost always are present in substoichiometric quantities. In addition, these modifications are a bit difficult to tackle with mass spectrometry that is generally used for the analysis of post-translational modifications. We focused on some simple and frequently occurring sugars modifying the side-chains of Ser and Thr residues of serum proteins. We worked with bovine serum. We developed an enrichment method using a carbohydrate-binding protein (lectin). We characterized the glycopeptide mixtures utilizing a novel MS/MS technique, electron-transfer dissociation (ETD). For this purpose the softwer used for database searching also had to be optimized. We identified ~40 novel glycosylation sites, more than anybody ever assigned in a single study. O-glycosylation occurs within the cell too: a single GlcNAc is deposited on Ser/Thr side chains. It fulfills a regulatory, signaling function. Though biologically very distant from its extracellular relatives, it represents a similar analytical challenge. We developed an enrichment method for this modification too

Light piping driven photosynthesis in the soil: low-light adapted active photosynthetic apparatus in the under-soil hypocotyl segments of bean (Phaseolus vulgaris)

Photosynthetic activity was identified in the under-soil hypocotyl part of 14-day-old soil-grown bean plants (Phaseolus vulgaris L. cv. Magnum) cultivated in pots under natural light-dark cycles. Electron microscopic, proteomic and fluorescence kinetic and imaging methods were used to study the photosynthetic apparatus and its activity. Under-soil shoots at 0-2 cm soil depth featured chloroplasts with low grana and starch grains and with pigment-protein compositions similar to those of the above-soil green shoot parts. However, the relative amounts of photosystem II (PSII) supercomplexes were higher; in addition a PIP-type aquaporin protein was identified in the under-soil thylakoids. Chlorophyll-a fluorescence induction measurements showed that the above- and under-soil hypocotyl segments had similar photochemical yields at low (10-55 µmol photons m-2 s-1) light intensities. However, at higher photon flux densities the electron transport rate decreased in the under-soil shoot parts due to inactivation of the PSII reaction centers. These properties show the development of a low-light adapted photosynthetic apparatus driven by light piping of the above-soil shoot. The results of this paper demonstrate that the classic model assigning source and sink functions to above- and under-soil tissues is to be refined, and a low-light adapted photosynthetic apparatus in under-soil bean hypocotyls is capable of contributing to its own carbon supply

Hydrolytic Mechanism of a Metalloenzyme Is Modified by the Nature of the Coordinated Metal Ion

The nuclease domain of colicin E7 cleaves double-strand DNA non-specifically. Zn2+ ion was shown to be coordinated by the purified NColE7 as its native metal ion. Here, we study the structural and catalytic aspects of the interaction with Ni2+, Cu2+ and Cd2+ non-endogenous metal ions and the consequences of their competition with Zn2+ ions, using circular dichroism spectroscopy and intact protein mass spectrometry. An R447G mutant exerting decreased activity allowed for the detection of nuclease action against pUC119 plasmid DNA via agarose gel electrophoresis in the presence of comparable metal ion concentrations. It was shown that all of the added metal ions could bind to the apoprotein, resulting in a minor secondary structure change, but drastically shifting the charge distribution of the protein. Zn2+ ions could not be replaced by Ni2+, Cu2+ and Cd2+. The nuclease activity of the Ni2+-bound enzyme was extremely high in comparison with the other metal-bound forms, and could not be inhibited by the excess of Ni2+ ions. At the same time, this activity was significantly decreased in the presence of equivalent Zn2+, independent of the order of addition of each component of the mixture. We concluded that the Ni2+ ions promoted the DNA cleavage of the enzyme through a more efficient mechanism than the native Zn2+ ions, as they directly generate the nucleophilic OH− ion

Polyploid Adipose Stem Cells Shift the Balance of IGF1/IGFBP2 to Promote the Growth of Breast Cancer

Background: The close proximity of adipose tissue and mammary epithelium predispose involvement of adipose cells in breast cancer development. Adipose-tissue stem cells (ASCs) contribute to tumor stroma and promote growth of cancer cells. In our previous study, we have shown that murine ASCs, which undergo polyploidization during their prolonged in vitro culturing, enhanced the proliferation of 4T1 murine breast cancer cells in IGF1 dependent manner. Aims: In the present study, our aim was to clarify the regulation of ASC-derived IGF1. Methods: 4T1 murine breast carcinoma cells were co-transplanted with visceral fat-derived ASCs (vASC) or with the polyploid ASC.B6 cell line into female BALB/c mice and tumor growth and lung metastasis were monitored. The conditioned media of vASCs and ASC.B6 cells were subjected to LC-MS/MS analysis and the production of IGFBP2 was verified by Western blotting. The regulatory effect was examined by adding recombinant IGFBP2 to the co-culture of ASC.B6 and 4T1. Akt/protein kinase B (PKB) activation was detected by Western blotting. Results: Polyploid ASCs promoted the tumor growth and metastasis more potently than vASCs with normal karyotype. vASCs produced the IGF1 regulator IGFBP2, which inhibited proliferation of 4T1 cells. Downregulation of IGFBP2 by polyploidization of ASCs and enhanced secretion of IGF1 allowed survival signaling in 4T1 cells, leading to Akt phosphorylation. Conclusions: Our results implicate that ASCs in the tumor microenvironment actively regulate the growth of breast cancer cells through the IGF/IGFBP system

Neuropeptidek radioaktív jelölése = Radioactive labelling of neuropeptides

Új jelölési módszereket fejlesztettünk ki neuropeptidek tríciummal való jelölésére. A módszerekhez új aminosavak és új neuropeptid prekurzorokra volt szükségünk. Aliciklikus béta-aminosavakat és ezek telítetlen analógjait szintetizáltunk vagy vásároltunk, racém vagy optikailag aktív formában. Az új aminosavakat felhasználtuk prekurzor peptidek szintéziséhez. A diasztereomer peptidek elválasztása radioaktív vagy nem radioaktív formában HPLC-vel történt. A radiojelölés általában trícium gázzal kettős kötés telítésével vagy dehalogénezéssel történt Pd katalizátorok jelenlétében. Az új módszerek között van a diszulfid kötést tartalmazó peptidek előállítása, valamint alaninban és valinban történő jelölés. Az aliciklikus béta-aminosavak radioaktív jelölése az első alkalommal valósult meg a jelöléstechnikában így ez a módszerünk úttörőnek tekinthető. Az így nyert radioaktív peptidek sokkal stabilabbak mint a természetes peptidek a biológiai rendszerekben. A 13 új radioaktív peptidünk (opioid peptidek, Substance P (1-7), Arg-vazopresszin) közül 3 peptidünk (Try-3H-ACPC-Phe-Phe-NH2, Dmt-3H-Pro-Phe-Phe-NH2, 3HDmt-DArg-Phe-Lys-NH2) kereskedelmi termék lett, ezen túlmenően radioaktív peptidjeink nemzetközi kutatási együttműködéseket és nemzetközi pályázatot is eredményezett. | Novel tritium labeling methods were developed for labeling of neuropeptides. New unnatural amino acids and neuropeptide precursors were needed for the labeling methods. Alicyclic-beta-amino acids and their analogues with unsaturated bond were synthesized or bought in raceme or optical pure forms. The new amino acids were used to the synthesis of the precursor peptides. The radio labeling occurred with tritium gas using catalytic saturation or dehalogenation methods using Pd catalyst. The diastereomer peptides in radioactive or no radioactive form were separated by HPLC. Among the new methods was the preparation of peptide containing disulphide bridge and radioactive labeling of peptides where the labels were in the alanine or valine residue. Pioneer method was developed when the label was introduced into the alicyclic beta-amino acids. These peptides were more stable than natural peptides in biological systems. Our 3 radioactive peptides (Dmt-DArg-Phe-Lys-NH2, Dmt-Pro-Phe-Phe-NH2, Tyr-Acpc-Phe-Phe-NH2) among 13 new peptides (opioid peptides, Substance P (1-7, 8Arg-vasopressin) became commercial product . Our radioactive peptides resulted in international research cooperation and international project